The octopamine receptor OAα1 influences oogenesis and reproductive performance in Rhodnius prolixus

The control of reproductive processes in Rhodnius prolixus involves a variety of neuroactive chemicals. Among these, several studies have suggested that the biogenic amine octopamine (OA), might play an active role in these processes. Here, we investigate the molecular profile of the R. prolixus α adrenergic-like OA receptor 1 (RpOAα1-R) and its role in egg production. Comparative molecular analyses confirm that the RpOAα1-R gene codes for a true OAα1 receptor. The RpOAα1-R transcript is highly expressed in tissues associated with egg production, and after a blood meal, which is the stimulus for full egg production in R. prolixus, the RpOAα1-R transcript is upregulated in the ovaries and spermatheca. After RNAi-mediated RpOAα1-R knockdown, an ovarian phenotype characterized by slow egg development is observed. Furthermore, an altered egg phenotype has been characterized with eggs that are deformed. Interestingly, there is no evidence of disruption in vitellogenin (Vg) synthesis by the fat body or uptake by the oocytes. On the other hand, RpOAα1-R downregulation is correlated with defective choriogenesis in the eggs. These results provide critical information concerning the role of OAα1-R in oogenesis in R. prolixus.


Introduction
Rhodnius prolixus, also known as the kissing bug, is one of the major vectors of the Chagas disease parasite Trypanosoma cruzi [1,2].R. prolixus is an obligate blood feeder and requires nutrients from a blood meal to undergo growth, development, and reproduction.In the adult female, a blood meal triggers neuroendocrinological events that result in a cascade of intracellular pathways associated with egg development [2].Among these, are pathways mediated by two hormones; juvenile hormone (JH) and 20-hydroxyecdysone (20E) [2][3][4][5][6].However, other neurochemicals are also involved in R. prolixus reproductive processes, including biogenic amines [7][8][9].
Biogenic amines are evolutionarily conserved molecules that function in neuro-signaling and play crucial roles in several physiological functions.Most biogenic amines are common to both vertebrates and invertebrates (dopamine, serotonin, and histamine), while some others are preferentially synthesized in either vertebrates, such as epinephrine and norepinephrine, or in invertebrates, such as tyramine (TA) and octopamine (OA) [10].OA is an important neurochemical in insects, synthesized from TA by the enzyme tyramine β-hydroxylase (TβH) [11].OA acts as a neurotransmitter, neuromodulator, and neurohormone controlling several physiological and behavioral aspects in insects such as locomotion, courtship, memory, and reproduction [12][13][14][15].OA exerts its effects through binding to specific G-protein coupled receptors (GPCRs), resulting in changes in second messenger levels, such as increasing intracellular Ca 2+ or altering cAMP levels [14].Based on their structure and intracellular pathway activated, OA receptors are classified into two α adrenergic-like OA receptors (OAα1-R, OAα2-R) and three β adrenergic-like OA receptors (OAβ1-R, OAβ2-R, OAβ3-R) [16].
OA signaling is well-known to be involved in insect reproduction [17].Both OA and OA receptors control and modulate physiological processes directly related to oogenesis and oviposition.In several insect species, R. prolixus included, OA modulates the myogenic activity of a variety of reproductive muscles, including those associated with the ovaries, oviduct, and bursa.For example, OA reduces the amplitude and frequency of oviduct contractions in Locusta migratoria [18], Drosophila melanogaster [19], and R. prolixus [7].In R. prolixus OA decreases spontaneous contractions of the oviducts and bursa and this action is mediated via increases in cAMP [7].Interestingly, the absence of OA, caused by TβH knockdown, has been linked with sterility in D. melanogaster [20][21][22], a dramatic phenotype rescued by OA ingestion [23].These physiological effects are linked to two OA receptors: OAα1-R (termed octopamine receptor in mushroom bodies, OAMB in D. melanogaster) and OAβ2-R.In D. melanogaster, the knockdown of OAMB causes female sterility due to egg retention [24,25].Deady & Sun [26] found that OAMB is necessary for follicle rupture during egg development in D. melanogaster.Moreover, an OAMB-mediated intracellular pathway has been linked with the proliferation of the female germinal stem cells in D. melanogaster [27].OAβ2-R is also needed for ovulation with a D. melanogaster OAβ2-R mutant being defective in egg-laying [28].Egg retention in ovaries has also been observed in Nilaparvata lugens females lacking OAβ2-R [29].The β-adrenergic receptor antagonist phentolamine blocks the inhibitory effects of OA on R. prolixus oviducts [7], and so taken together, OA may modulate oviduct musculature via OAβ2-R.
The involvement of OAα1-R in R. prolixus reproduction has not been reported, and so this paper examines the possible role of OAα1-R in egg production, particularly oogenesis, egg laying, and hatching in R. prolixus.

R. prolixus rearing
Adult females of R. prolixus were taken from an established colony at the University of Toronto Mississauga and reared in incubators at 25˚C under high humidity (*50%).Adult females at 7 days post-ecdysis (d PE) were fed through an artificial feeding membrane on defibrinated rabbit blood (Cedarlane Laboratories Inc., Burlington, ON, Canada) in order to stimulate normal egg production.Adult females post blood meal (PBM) were placed individually in a cage with two fed males for copulation.

Isolation and cloning of full-length RpOAα1-R
Total RNA was extracted from the reproductive system of adult R. prolixus females using TRI-zol™ Reagent according to the manufacturer's protocol (Thermo Fisher Scientific, Waltham, Massachusetts, USA), quantified in a spectrophotometer DS-11+ (DeNovix Inc., Wilmington, DE, USA) and analyzed by 1% w/v agarose gel electrophoresis.One μg of RNA was treated with DNase I (Thermo Fisher Scientific) and used for the synthesis of cDNA, carried out with SMARTer RACE 3'/5' Kit (Takara, Japan) according to the manufacturer's instructions.To identify the full R. prolixus OAα1 open reading frame (ORF), a blast search on VectorBase was performed using Nilaparvata lugens (XM_039440532.1) and Cimex lectularius (XM_024229855.1)OAα1-R sequences as templates.Two specific reverse primers (S1 Table ) were designed for the amplification of the 5 0 cDNA end of the receptor (5 0 Modified RACE) using SMARTer RACE 3'/5' Kit (Takara, Japan).The final PCR product corresponding to the complete R. prolixus OAα1-R ORF was amplified by specific primers (S1 Table ), gel purified, cloned into pJET 1.2/blunt vector (Thermo Fisher Scientific) and transformed into Escherichia coli XL-1 chemically competent cells.Positive clones were selected using LB broth agar plates with 100 μg ml −1 ampicillin.Plasmid was then extracted by EZ-10 spin column plasmid DNA miniprep kit (Bio Basic, Ontario, Canada) and verified by DNA sequencing (Macrogen, Seul, South Korea).The sequence, named RpOAα1-R, is deposited in GenBank with the accession number (OR248009).

RNA extraction and RT-qPCR analysis
The RpOAα1-R expression profile was investigated in the central nervous system (CNS), fat body, bursa, oviducts, spermatheca, tropharium, and follicles of 7 d post ecdysis (PE) unfed adult females.In addition, RpOAα1-R mRNA was analyzed throughout different time points PBM (1, 3, and 5 d) in the same tissues as above.For this purpose, 7 d PE adult R. prolixus females were fed with rabbit blood, and tissues collected starting from 1 d PBM.The individual tissues from three animals were dissected and pooled for each biological replicate.In order to investigate the expression profile of JH and 20E pathway actors, fat body and ovaries were dissected from 6 d PBM females, treated with dsOAα1-R or control dsRNA.Total RNA was extracted using TRIzol™ Reagent according to the manufacturer's protocol (Thermo Fisher Scientific, Waltham, Massachusetts, USA).Five hundred ng of purified RNA was then treated with Turbo DNase I (Invitrogen, Waltham, Massachusetts, USA) and used for cDNA synthesis, carried out with High-Capacity cDNA Reverse Transcription kit (Applied Biosystem, Waltham, Massachusetts, USA) according to the manufacturer's instructions.qPCR was performed using a 384-CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, California, USA) in a 10 μL reaction mixture containing 4.2 μL of cDNA previously diluted 1:5, 5 μL of Advanced qPCR Master Mix (Wisent Bioproducts Inc., Saint-Jean-Baptiste, QC, Canada), 0.4 μL forward primer (10 μmol l −1 ), and 0.4 μL reverse primer (10 μmol l −1 ).Thermal cycling conditions were 95˚C for 3 min, 40 cycles at 95˚C for 15 s, and 60˚C for 30 s.After the cycling protocol, a melting-curve analysis from 65 to 95˚C was applied.mRNA abundance of the analyzed genes was normalized by the 2 -ΔΔCt method [30] or the relative quantification method [31] using Actin and Rp49 [32] primers (S1 Table ).Each biological sample was performed having two technical replicates and using no-template controls.

Fluorescent in situ hybridization (FISH)
The fluorescent in situ hybridization (FISH) technique was used to determine the expression pattern of RpOAα1-R in the female reproductive system.The OAα1-R fragment amplification was performed by using antisense gene-specific primers containing the T7 RNA polymerase promoter sequence (S1 Table ).Then, the DIG RNA labeling kit SP6/T7 (Roche Applied Science, Mannheim, Germany) was used in order to synthesize the digoxigenin (DIG)-labeled antisense RNA probes.Hybridization was done by incubating the reproductive system from unfed R. prolixus females overnight, at 4˚C, with 100 ng (final concentration: 1 ng/μL) of the specific probe.The signal specificity of the antisense RNA probe was tested on insects treated with dsOAα1-R.Then, a biotin-SP-conjugated monoclonal mouse anti-digoxigenin antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) was incubated (1:400) overnight at 4˚C with the tissues, under agitation.The tissues were then washed and incubated in 1:100 horseradish peroxidase-streptavidin solution (Molecular Probes, Eugene, OR, USA).For signal amplification, tissues were incubated with Alexa Fluor 568 tyramide solution (Molecular Probes, Eugene, OR, USA).Finally, tissues were mounted on slides with one drop of Fluoroshield (Sigma-Aldrich, ON, Canada).A confocal microscope LSM-800 (Carl Zeiss, Jena, Germany) with the Zeiss LSM Image Browser software was used to obtain and analyse the pictures.

Double-stranded RNA design and synthesis
BlasTaq™ 2X PCR Taq MasterMix (ABM, Vancouver, BC, Canada) and primers with 5' extensions containing T7 promoters (S1 Table ) were used to generate the R. prolixus dsRNA for RpOAα1-R and the control dsRNA (ampicillin resistance gene or ARG) amplicons, 350-500 bp long.Then, the in vitro dsRNA synthesis was performed by T7 RiboMAX™ Express Large Scale RNA Production System (PROMEGA, Madison, Wisconsin, USA).The RpOAα1-R sequence used to synthesize the dsRNA is shown in S1 Fig.

Knockdown of transcript expression using double-stranded RNA
RpOAα1-R downregulation was achieved by injecting 7 d PE R. prolixus adult females with 5 μL (1 μg/μL) of specific dsRNA using a 10 μL Hamilton 80330 standard microliter syringe (Reno, Nevada, USA).The access to a blood meal was given 7 days after the injection.Since the amount of blood ingested affects the number of eggs laid, insects were weighed before and after the blood meal.The RNAi silencing efficiency was evaluated by RT-qPCR on the whole reproductive system from unfed insects 7 days PI, and 6 days PBM (13 days PI).

Egg laying and hatching ratio analysis
The number of eggs laid by dsARG (control) and dsOAα1-injected R. prolixus adult females was recorded over 20 d PBM.Fifteen insects were used to monitor egg laying in each treatment.For the hatching ratio, eggs from control or dsOAα1-injected females were collected and 14 days after the first egg hatched the number of nymphs alive was recorded.

Chorion protein extraction and SDS-PAGE
Fifteen eggs from dsOAα1-R (both phenotypes) and dsARG females were opened under a stereomicroscope in cold 0.01M Tris/HCl pH 8.4 to remove the yolk protein content.The chorions were then carefully washed, three times, in 0.01 M Tris/HCl pH 8.4.Then, the chorions were treated as previously described by Santos & Ramos [33].Briefly, the chorions were homogenized in 8 M urea, 360 mM Tris/HCl (pH 8.4), and 30 mM dithiothreitol using a manual pestle and incubated at room temperature for 30 min.The samples were centrifuged at 12,000 g for 10 min, and the supernatant was collected.15 μl of the urea-extracted proteins were loaded into a pre-made gel (percentage 4-20%, Mini-Protean TGX Stain-Free Precast Gels, Bio-Rad, Hercules, California, USA) and stained with Coomassie.Densitometry was performed using ImageJ.

Statistical analysis
Graph Pad Prism 9.0 (La Jolla, California, USA) was used to process all the data presented in the article.All data represent means ± s.e.m., evaluated using one-way ANOVA followed by Dunnett's test for multiple comparisons, or Student's T test, or F test for comparing cumulative egg laying over time.

Molecular characterization of RpOAα1-R
The open reading frame sequence of RpOAα1-R is 1866 bp long and codes for a 621 amino acid polypeptide with a predicted MW of 68 kDa (S2 Fig) .In terms of structural domains, TMHMM v.2.0 software suggests seven putative transmembrane domains, as expected for a GPCR (S3 Fig) .The helixes are flanked by an extracellular N-terminus of 30 residues and a long intracellular C-terminus of 80 residues.Furthermore, the RpOAα1-R sequence contains a DRY conserved sequence just after transmembrane (TM) III, one N-glycosylation site in the extracellular N-terminus, and twelve P-glycosylation sites, two for PKA and ten for PKC (S2 Table ).Moreover, at position 107 in TM III there is a conserved aspartic acid (D107) responsible for the interaction with OA and TA, the endogenous agonists of the receptor (Huang et al., 2007)

(S2 Fig).
The RpOAα1-R amino acid sequence was then compared with that of several OA and TA receptors, allowing the construction of a neighbor-joining phylogenetic tree using MEGA 11 (Fig 1).As expected, RpOAα1-R grouped with the OA α-adrenergic-like family and shares the highest percentage identity with the Nilaparvata lugens OA α-adrenergic-like receptor (GenBank accession no.XM_039440532.1),another Hemipteran.Based on the phylogenetic results, a multiple sequence alignment was performed between the RpOAα1-R amino acid sequence and other insect OAα1 receptors (S4 Fig) .The analysis further confirms the similarity of RpOAα1-R to known OAα1 receptors, showing the typical GPCR structure with highly conserved domains corresponding to the transmembrane regions.

RpOAα1-R is expressed in reproductive tissues in unfed and fed adult R. prolixus females
To investigate the putative role of OAα1-R in R. prolixus reproduction, transcript expression of RpOAα1-R was quantified in R. prolixus tissues from both unfed and fed adult females (CNS, fat body, bursa, oviducts, spermatheca, follicles, and tropharium) (Fig 2).In unfed insects, RpOAα1-R is mostly expressed in the CNS and antennae, and in reproductive tissues such as the spermatheca and the ovariole structures, the follicles and tropharium (Fig 2A).After a blood meal, upregulation of RpOAα1-R expression is observed in the fat body, bursa,

RpOAα1-R specific transcript expression in female R. prolixus reproductive tissues by fluorescent in situ hybridization
To confirm the RpOAα1-R mRNA enrichment observed in the reproductive system by RT-qPCR, we examined the region-specific expression of the receptor in the reproductive system of unfed adult R. prolixus females by FISH (Fig 3A).Specific RpOAα1-R mRNA fluorescent signal is detected in the tropharium, follicles, and spermatheca, with a lower or no signal in the oviducts and bursa (Fig 3B -3E).Moreover, a specific signal for RpOAα1-R has been identified in the follicle cells and within the cytoplasm of the oocytes, as shown in the

RpOAα1-R signaling is essential for normal oogenesis
To investigate the role of RpOAα1-R in the reproductive physiology of R. prolixus adult females, dsRNA designed to specifically target the RpOAα1-R receptor sequence was injected into 7 d PE virgin adult females (Fig 4A) which were then fed and mated 7 d post-injection (PI) and tissue collected at 6 d PBM.To check for any potential effects of dsRNA injection on feeding (in terms of the amount of blood ingested), the injected insects were weighed before   the abnormal eggs have a reduced total protein content (Fig 5C).In addition to the altered hatching ratio and the lower protein content, abnormal eggs exhibit less amount of chorion proteins compared to the controls and to the normal eggs from dsOAα1-R injected females.Indeed, the chorion protein Rp45 is significantly reduced in the abnormal eggs laid from females with RpOAα1-R downregulated (Fig 5E and 5F).Furthermore, the transcripts for Rp30 and Rp45 are decreased in the ovaries from insects treated with dsOAα1-R, with Rp45 statistically downregulated, suggesting a defective synthesis and/or accumulation of the chorion proteins compared to the controls (Fig 5F).

Juvenile hormone (JH) and 20-hydroxyecdysone (20E) pathways are not involved in alterations in oogenesis induced by dsRpOAα1-R
Since JH signaling is directly involved in R. prolixus yolk protein synthesis [2,34] and 20E pathways have been recently correlated with R. prolixus oogenesis [6,35,36], their possible involvement in the altered egg phenotype after dsOAα1-R injection was investigated by RT-qPCR.To address this, the expression profile of proteins transcribed after the JH pathway is activated (vitellogenin genes Vg1 and Vg2, Krüppel homolog1 Kr-h1 and the vitellogenin receptor VgR) and the 20E pathway is activated (ultraspiracle protein USP, ecdysone receptor EcR and the Halloween genes Shadow and Shade) were explored.After dsOAα1-R injection, Kr-h1 and Vg1 and Vg2 in the fat body at 6 d PBM were not altered but Vg1 was downregulated by ~50% in the ovaries at 6

Discussion
Although localized in tropical areas, climate change is responsible for creating new territories where R. prolixus can become endemic [37].Moreover, R. prolixus is difficult to control by chemical approaches and the only way to treat Chagas disease is with two drugs that have limitations on treatment timing and serious side effects [38].Because of its reduced susceptibility to traditional control strategies, new methods for R. prolixus control are needed.Therefore, identifying novel chemical compounds and new biological targets based on R. prolixus physiology is crucial.This study deals with the molecular characterization of the R. prolixus OAα1 receptor (RpOAα1-R).Moreover, by applying RNAi-mediated knockdown of RpOAα1-R, it was possible to reveal the importance of the OA pathway in the reproductive process in R. prolixus.
The RpOAα1-R polypeptide shares many structural features with OAα1 receptors from other insects and GPCRs in general [39].RpOAα1-R contains seven highly conserved transmembrane segments, as expected for a GPCR, as well as phosphorylation and glycosylation sites, typical for this receptor class and essential for correct protein folding and receptor signaling [40,41].Concerning the OA binding site, the main amino acid residue interacting with the endogenous agonist is an aspartic acid (Asp) located in TM III and well conserved in all the GPCRs [42].The artificial mutation of the Asp residue in Anopheles gambiae AgOAR45B mutant resulted in the loss of responsiveness to OA [43].Moreover, the mutant Bombyx mori OAα1-R D103A was unable to bind OA, indicating that the Asp residue is implicated in the binding and activation of the receptor by OA [44].Therefore, it is mostly likely that the Asp residue localized in position 103 (D103) in RpOAα1-R polypeptide sequence may be implicated in OA binding.However, the same two studies showed that different residues localized in TM V and VI might also be involved in binding with OA, possibly revealing a more complex ligand/receptor-coupling mechanism [43,44].
To further investigate the role that RpOAα1-R might play in R. prolixus reproduction, an expression profile analysis of the receptor was performed.RT-qPCR on unfed R. prolixus adult females revealed that RpOAα1-R mRNA is enriched especially in the CNS compared to the other tissues analyzed.Since OA is one of the most important neurotransmitters in invertebrates, it is not surprising that the RpOAα1-R transcript is enriched in the CNS [14].This is in keeping with that observed in other insects, including Aedes aegypti, A. gambiae, D. melanogaster, Laodelphax striatellus, and Apis mellifera [43,[45][46][47][48][49].Perhaps not surprisingly, RpOAα1-R levels are higher in the antennae of unfed R. prolixus females, probably given its well-established role in insect olfaction [50].In line with this hypothesis, in A. mellifera and D. melanogaster, OAα1-R is found highly expressed in the antennal lobes, strongly suggesting a role in modulating olfactory responses [51].Antennae are organs also responsible for sound perception, since OA has an important role in modulating sound responses in other blood-feeding insects (i.e., A. gambiae; [52][53][54]).Therefore, it might be possible that the enrichment of the RpOAα1-R transcript in the antennae reflects its possible role in hearing in R. prolixus.
In addition to the CNS and antennae, RpOAα1-R mRNA is enriched in the reproductive system, including the spermatheca, follicles, and tropharium; the latter two structures being part of the ovarioles of R. prolixus [2].Interestingly the RpOAα1-R mRNA is not upregulated after a blood meal in the spermathecal or tropharium but is so in the follicles.OA may play a role in sperm storage/release in the spermatheca and maintenance in the tropharium that contains germ cells and nurse cells.The same receptor is important for reproductive success in other insects, including D. melanogaster and L. striatellus [27,49,55].In D. melanogaster, OAMB modulates contraction of the oviduct and bursa after OA activation [22,26,56].Furthermore, OAα1-R controls ovulation by modulating follicle rupture and consequently the release of the egg [55].After a blood meal, RpOAα1-R expression levels are significantly increased in the fat body, bursa, oviduct, and follicles strongly suggesting that the receptor may play an important role in R. prolixus reproduction.This was confirmed by RNAi-mediated RpOAα1-R knockdown which significantly affects R. prolixus oogenesis, egg laying, and hatching.
Interestingly, the increase in RpOAα1-R expression after a blood meal is not immediate, but in most tissues, such as the oviduct and bursa, occurs 5 d PBM.We cannot exclude the possibility that the "late" increase in RpOAα1-R expression in the bursa and oviducts might be linked to the receptor's role in muscle modulation, and expression in the follicles to ovulation.However, after RpOAα1-R downregulation, we did not observe any specific phenotypes, such as egg retention in the ovaries, oviduct or bursa and further experiments need to be performed in order to test these hypotheses.
Compared to the control group, fewer eggs are laid by R. prolixus dsOAα1-R-treated females.Moreover, 30% of the eggs laid are deformed resulting in hatching failure.In A. gambiae, the pharmacological alteration of the OAα1-R signaling by different OA agonist and/or antagonist clonidine, prazosin, and phenylephrine injection, affected egg development and egg laying [57].Egg deformation is observed also in L. striatellus after OAα1-R silencing by RNAi [49].In this case, the authors suggested that the altered egg development was due to an inhibited Vg synthesis occurring in the fat body upon OAα1-R downregulation.In our experiments, however, knockdown of RpOAα1-R did not alter JH or 20E pathways and, consequently, the synthesis of Vg in the fat body was comparable to that observed in the control group.Therefore, it is likely that the transcriptional increase of RpOAα1-R in the fat body PBM is not attributable to a direct role of the receptor in modulating Vg synthesis in the fat body but could be related to other nutrients or homeostatic processes.Furthermore, the expression levels of VgR in the ovaries, in both treated and control groups, were similar, indicating that no impairment in Vg uptake by ovaries occurred after RpOAα1-R downregulation.Vg1 synthesis appeared altered only in the ovaries after dsOAα1-R injection.However, it remains to be investigated whether the Vg1 transcript downregulation in the ovaries is directly affected by the RNAi-mediated RpOAα1-R downregulation or to a lesser protein demand from the defective eggs.Halloween genes essential for ecdysteroid synthesis, such as shadow and shade, play an important role in R. prolixus oogenesis [6,35], and there are profound effects on egg production and egg shape when ecdysteroid signaling is disrupted.However, after RNAi-mediated silencing of RpOAα1-R, the transcript expression levels of shadow and shade and the USP and EcR subunits were comparable between control and treated insect, suggesting that knockdown of RpOAα1-R did not alter expression of ecdysteroids.
A key step in oogenesis is the formation of the chorion, a process known as "choriogenesis".During this process, the mature oocyte is coated with newly synthesized layers of eggshell, which are assembled and cover the entire oocyte surface.After choriogenesis, the eggs are ready to be fertilized and laid onto a substrate in the environment [58].In R. prolixus, chorion formation is a complex multi-step process that requires different intracellular pathways, including autophagy [33,59].Investigating the chorion properties in dsOAα1-R-treated R. prolixus, we observed alterations in both transcript and protein content of two specific chorion protein, Rp30 and Rp45.In R. prolixus, both proteins are expressed during the final stage of vitellogenesis, by the follicular cells [60].Since follicles, during vitellogenesis, express higher transcript levels of RpOAα1-R, we cannot rule out the possibility of a direct OA-mediated pathway that modulates Rp30 and Rp45 synthesis during vitellogenesis in R. prolixus.However, since the cellular mechanisms that regulates Rp30 and Rp45 synthesis are not well characterized, this remains to be seen.It has recently been shown that the RNAi-mediated alteration of the ecdysone pathway in R. prolixus dramatically affected the synthesis of both Rp30 and Rp45 chorion proteins, producing abnormal eggs, as well as a reduction in number of eggs made and laid and their reduced hatching rate [6,35].It is therefore possible that OA, through the RpOAα1-R receptor, is also involved in chorion synthesis during oogenesis but appears to act independent of 20E and JH.

Conclusions
In conclusion, this study provides new insights on the molecular characterization and physiological function of the R. prolixus OAα1-R.Given its crucial role in R. prolixus reproduction, it could be considered as a new target for R. prolixus control.Further characterization of the OAα1 receptor, e.g. its pharmacological profile, is crucial to discover innovative control strategies targeting this octopamine receptor.

Fig 1 .
Fig 1. Phylogenetic relationship determined using maximum likelihood analysis of RpOAα1-R and other insect amine receptors.The characterized RpOAα1-R is outlined in red.The values shown at the nodes of the branches are the percentage bootstrap support (1000 replications) for each branch.Alignment was performed using the amino acid sequences found in GenBank (accession number for each sequence is noted and reported in S3 Table).D. melanogaster OR83b receptor was chosen as the outgroup.https://doi.org/10.1371/journal.pone.0296463.g001

Fig 3E .
This localization pattern reflects the expression profile of the RpOAα1-R transcript observed by RT-qPCR (Fig 2A).As a negative control, FISH experiments were performed on insects treated with dsOAα1-R to downregulate the receptor.As expected, lower or no labeling, specific for dsOAα1-R, is observed in either the spermatheca, tropharium or follicles (S5 Fig).

Fig 3 .
Fig 3. Fluorescent in situ hybridization analysis of RpOAα1-R transcript in the reproductive system of unfed R. prolixus adult females.Anatomy of the female R. prolixus reproductive system (A).Localization of the RpOAα1-R transcript across the reproductive system.CO: common oviduct; LO: lateral oviduct (B).Localization of the RpOAα1-R transcript in the spermatheca (C), tropharium (D), and follicle (E).FISH experiments were repeated twice, using 3 reproductive systems in each replicate.https://doi.org/10.1371/journal.pone.0296463.g003

Fig 4 .
Fig 4. Effects of RNAi-mediated RpOAα1-R knockdown on R. prolixus oogenesis and egg laying.Experimental approach (A).Weight of dsRNA-injected insects following a blood meal.Data are the means ± sem of 13 independent replicates (each replicate composed of 1 female, and shown as individual data points) (B).The reproductive system was dissected 7 and 13 d post-injection (PI) to measure RNAi efficacy of RpOAα1-R transcript knockdown.The results are shown as the mean ± sem (n = 6, where each n represents tissue from 1 insect).Statistically significant differences were determined by Student's t-test *p < 0.05; **p < 0.01 (C).The representative phenotype of the reproductive systems of adult R. prolixus females injected with dsARG or dsOAα1-R 13 d PI (equivalent to 6 d PBM) (D).Cumulative number of eggs laid per female injected with dsARG or dsOAα1-R over 15 d PBM.The results are shown as the mean ± sem (n = 15 insects).The statistical difference between linear regression slopes was performed by F-test (P = 0.0001) (E).Cumulative average of eggs laid per female by 15 d PBM.Statistical significance was determined by a Student's t-test *p < 0.05 (F).https://doi.org/10.1371/journal.pone.0296463.g004

Fig 5 .
Fig 5. RpOAα1-R downregulation causes chorion-defects and unviable eggs.Representative images of dsARG and dsOAα1-R treated insect eggs.Scale bar: 1 mm (A).Hatching ratio throughout 14 days after the first egg hatched (n = 3, each n represents 15 eggs, shown as individual data points) (B).Total protein in eggs from R. prolixus females injected with dsARG or dsOAα1-R (n = 5 shown as individual data points, with each n composed of 3 eggs, 5 to 7 days after oviposition) (C).4-20% SDS-PAGE of the urea-extracted proteins from control and dsOAα1-R egg chorions, collected 5 to 7 days after oviposition.Arrowheads indicate chorion proteins Rp30, Rp45, and Rp200 (D).The uncropped gel picture is available as S6 Fig. Densitometry analysis of the chorion proteins indicated in (D) performed using Image J. Graph shows mean ± sem (n = 3, shown as individual data points) (E).Effect of dsOAα1-R treatment on the chorion proteins Rp30 and Rp45 expression profile in the ovaries (n = 5).*p < 0.05 according to Student's t-test (F).https://doi.org/10.1371/journal.pone.0296463.g005 d PBM with no change in Vg2 and VgR (Fig 6A).Concerning the 20E pathway, USP, EcR and the Halloween genes Shade and Shadow are not altered at in their expression profile at 6 d PBM compared to the controls (Fig 6B).Taken together, these data suggest that the effects of RNAi-mediated RpOAα1-R silencing do not involve JH or 20E in R. prolixus.

Fig 6 .
Fig 6.RT-qPCR analysis of JH and 20E pathway elements in the fat body and ovary of adult, 6 d PBM, mated R. prolixus females after dsOAα1-R treatment.RNAs were isolated from the fat body and ovary of dsARG (control) or dsOAα1-R injected R. prolixus females.RT-qPCR of mRNA expression of JH pathway elements Vg1, Vg2, and Kr-h1 in the fat body, and Vg1, Vg2, and VgR in the ovaries (A).RT-qPCR of mRNA expression of 20E pathway elements USP, EcR, Shadow, and Shade in the ovaries (B).Actin and Rp49 were used as reference genes.Data are mean ± sem of 5 independent replicates (each composed of tissues from 1 insect).Statistical analysis was performed using Student's t-test.*p < 0.05.https://doi.org/10.1371/journal.pone.0296463.g006